A Novel Motif in the 3'-UTR of PRRSV-2 Is Critical for Viral Multiplication and Contributes to Enhanced Replication Ability of Highly Pathogenic or L1 PRRSV.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) with enhanced replication capability emerged in China and has become dominant epidemic strain since 2006. Up to now, the replication-regulated genes of PRRSV have not been fully clarified. Here, by swapping the genes or elements between HP-PRRSV and classical PRRSV based on infectious clones, NSP1, NSP2, NSP7, NSP9 and 3'-UTR are found to contribute to the high replication efficiency of HP-PRRSV. Further study revealed that mutations at positions 117th or 119th in the 3'-UTR are significantly related to replication efficiency, and the nucleotide at position 120th is critical for viral rescue. The motif composed by 117-120th nucleotides was quite conservative within each lineage of PRRSV; mutations in the motif of HP-PRRSV and currently epidemic lineage 1 (L1) PRRSV showed higher synthesis ability of viral negative genomic RNA, suggesting that those mutations were beneficial for viral replication. RNA structure analysis revealed that this motif maybe involved into a pseudoknot in the 3'-UTR. The results discovered a novel motif, 117-120th nucleotide in the 3'-UTR, that is critical for replication of PRRSV-2, and mutations in the motif contribute to the enhanced replicative ability of HP-PRRSV or L1 PRRSV. Our findings will help to understand the molecular basis of PRRSV replication and find the potential factors resulting in an epidemic strain of PRRSV.

Synergistic Pathogenicity by Coinfection and Sequential Infection with NADC30-like PRRSV and PCV2 in Post-Weaned Pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCVs) are two major viruses that affect pigs. Coinfections between PRRSV and PCV2 are frequently reported in most outbreaks, with clinical presentations involving dyspnea, fever, reduced feed intake, weight loss, and death in fattening pigs. The NADC30-like PRRSV and PCV2d are the main circulating virus strains found in China. This study determines the impact of NADC30-like PRRSV and PCV2d mono-infection and coinfection on the immune system, organ pathology, and viral shedding in five-week-old post-weaned pigs. Pigs were randomly divided into six groups: PBS, PRRSV, PCV2, PRRSV-PCV2 coinfection (co), and PRRSV-PCV2 or PCV2-PRRSV sequential infections. Fever, dyspnea, decreased feed intake, weight loss, and pig deaths occurred in groups infected with PRRSV, Co-PRRSV-PCV2, and PRRSV-PCV2. The viral load was higher in Co-PRRSV-PCV2, PRRSV-PCV2, and PCV2-PRRSV than those mono-infected with PRRSV or PCV2. Additionally, cytokines (IFN-γ, TNF-α, IL-4, and IL-10) produced by pigs under Co-PRRSV-PCV2 and PRRSV-PCV2 groups were more intense than the other groups. Necropsy findings showed hemorrhage, emphysema, and pulmonary adhesions in the lungs of pigs infected with PRRSV. Smaller alveoli and widened lung interstitium were found in the Co-PRRSV-PCV2 and PRRSV-PCV2 groups. In conclusion, PRRSV and PCV2 coinfection and sequential infection significantly increased viral pathogenicity and cytokine responses, resulting in severe clinical signs, lung pathology, and death.

Identification of Cryptic Promoter Activity in cDNA Sequences Corresponding to PRRSV 5' Untranslated Region and Transcription Regulatory Sequences.

To investigate the role of PRRSV nonstructural proteins (nsps) in viral RNA replication and transcription, we generated a cDNA clone of PRRSV strain NCV1 carrying the nanoluciferase (nluc) gene under the control of the transcription regulatory sequence 6 (TRS6) designated as pNCV1-Nluc. Cells transfected with the pNCV1-Nluc DNA plasmid produced an infectious virus and high levels of luciferase activity. Interestingly, cells transfected with mutant pNCV1-Nluc constructs carrying deletions in nsp7 or nsp9 regions also exhibited luciferase activity, although no infectious virus was produced. Further investigation revealed that the cDNA sequences corresponding to the PRRSV 5' untranslated region (UTR) and TRS, when cloned upstream of the reporter gene nluc, were able to drive the expression of the reporter genes in the transfected cells. Luciferase signals from cells transfected with a reporter plasmid carrying PRRSV 5' UTR or TRS sequences upstream of nluc were in the range of 6- to 10-fold higher compared to cells transfected with an empty plasmid carrying nluc only. The results suggest that PRRSV 5' UTR and TRS-B in their cDNA forms possess cryptic eukaryotic promoter activity.

Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) genetic diversity and occurrence of wild type and vaccine-like strains in the United States swine industry.

Porcine reproductive and respiratory syndrome virus genotype 2 (PRRSV-2) genetic diversity in the U.S. was assessed using a database comprising 10 years' worth of sequence data obtained from swine production systems routine monitoring and outbreak investigations. A total of 26,831 ORF5 PRRSV-2 sequences from 34 production systems were included in this analysis. Within group mean genetic distance (i.e. mean proportion of nucleotide differences within ORF5) per year according to herd type was calculated for all PRRSV-2 sequences. The percent nucleotide difference between each sequence and the ORF5 sequences from four commercially available PRRSV-2 vaccines (Ingelvac PRRS MLV, Ingelvac PRRS ATP, Fostera PRRS, and Prevacent PRRS) within the same lineage over time was used to classify sequences in wild-type or vaccine-like. The mean ORF5 genetic distance fluctuated from 0.09 to 0.13, being generally smaller in years in which there was a relative higher frequency of dominant lineage. Vaccine-like sequences comprised about one fourth of sequences obtained through routine monitoring of PRRS. We found that lineage 5 sequences were mostly Ingelvac PRRS MLV-like. Lineage 8 sequences up to 2011 were 62.9% Ingelvac PRRS ATP-like while the remaining were wild-type viruses. From 2012 onwards, 51.9% of lineage 8 sequences were Ingelvac PRRS ATP-like, 45.0% were Fostera PRRS-like, and only 3.2% were wild-type. For lineage 1 sequences, 0.1% and 1.7% of the sequences were Prevacent PRRS-like in 2009-2018 and 2019, respectively. These results suggest that repeated introductions of vaccine-like viruses through use of modified live vaccines might decrease within-lineage viral diversity as vaccine-like strains become more prevalent. Overall, this compilation of private data from routine monitoring provides valuable information on PRRSV viral diversity.

Pathogenicity of Porcine Circovirus Type 2d (PCV2d) in Pigs Infected with PCV2d or Co-infected with Mycoplasma hyopneumoniae and PCV2d or with Porcine Reproductive and Respiratory Syndrome Virus and PCV2d.

The objective of this study was to determine the pathogenicity of porcine circovirus type 2d (PCV2d) in pigs inoculated intranasally with PCV2d alone, PCV2d in combination with Mycoplasma hyopneumoniae or PCV2d in combination with porcine reproductive and respiratory syndrome virus (PRRSV). Pigs infected with PCV2d alone were asymptomatic. All pigs inoculated with either M. hyopneumoniae and PCV2d or with PCV2d and PRRSV developed porcine circovirus-associated disease (PCVAD), as characterized by a sudden onset of clinical signs and disseminated granulomatous inflammation. Inflammation was mainly present in lymph nodes and spleen, and occasionally in liver and kidney. Pigs in both of these dually infected groups also had significantly higher (P <0.05) microscopic lymphoid lesion scores and a significantly higher (P <0.05) number of PCV2-positive cells in lymph node tissue than did pigs inoculated with PCV2d alone. The M. hyopneumoniae and PRRSV combination potentiated the PCV2d load in the blood. Co-infection with PRRSV and PCV2d resulted in a significantly higher blood load of PCV2d compared with the M. hyopneumoniae and PCV2d combination. Successful reproduction of PCVAD in pigs appears to require PCV2d with at least one additional infectious agent, such as M. hyopneumoniae or PRRSV, for the full manifestation of disease.

A strain of highly pathogenic porcine reproductive and respiratory syndrome virus: genomic characterization, pathogenicity, and construction of an infectious full-length cDNA clone.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which inflicts major economic losses on the global pig farming industry. Based on its similarity to highly pathogenic strains, the GDzj strain isolated in this study was predicted to be highly pathogenic. We therefore analyzed the pathogenicity of this strain experimentally in piglets. All piglets challenged with this virus experienced fever or high fever, loss of appetite, decreased food intake, daily weight loss, shortness of breath, and listlessness, and the necropsy results showed that they had experienced severe interstitial pneumonia. We then used the BAC system to construct a full-length cDNA infectious clone of GDzj, and the rescued virus displayed in vitro proliferation characteristics similar to those of the parental PRRSV strain. In summary, we successfully isolated a highly pathogenic PRRSV strain and constructed a full-length infectious cDNA clone from it, thereby providing an effective reverse genetics platform for further study of viral pathogenesis.

Swine Dendritic Cell Response to Porcine Reproductive and Respiratory Syndrome Virus: An Update.

Dendritic cells (DCs) are the most potent antigen-presenting cells, unique to initiate and coordinate the adaptive immune response. In pigs, conventional DCs (cDCs), plasmacytoid DCs (pDCs), and monocyte-derived DCs (moDCs) have been described in blood and tissues. Different pathogens, such as viruses, could infect these cells, and in some cases, compromise their response. The understanding of the interaction between DCs and viruses is critical to comprehend viral immunopathological responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important respiratory pathogen in the global pig population. Different reports support the notion that PRRSV modulates pig immune response in addition to their genetic and antigenic variability. The interaction of PRRSV with DCs is a mostly unexplored area with conflicting results and lots of uncertainties. Among the scarce certainties, cDCs and pDCs are refractory to PRRSV infection in contrast to moDCs. Additionally, response of DCs to PRRSV can be different depending on the type of DCs and maybe is related to the virulence of the viral isolate. The precise impact of this virus-DC interaction upon the development of the specific immune response is not fully elucidated. The present review briefly summarizes and discusses the previous studies on the interaction of in vitro derived bone marrow (bm)- and moDCs, and in vivo isolated cDCs, pDCs, and moDCs with PRRSV1 and 2.

The jigsaw of PRRSV virulence.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of the, probably, most economically important disease for the pig industry worldwide. This disease, characterised by producing reproductive failure in sows and respiratory problems in growing pigs, appeared in the late 1980s in the United States and Canada. Since its appearance, strains capable of producing higher mortality rates as well as greater severity in clinical signs and lesions than classical strains have been identified. However, since the first reports of these "virulent" PRRSV outbreaks, no homogeneity and consensus in their description have been established. Moreover, to the authors' knowledge, there is no published information related to the criteria that a PRRSV strain should fulfil to be considered as a "virulent" strain. In this review, we revise the terminology used and gather the information related to the main characteristics and differences in clinical signs, lesions, viral replication and tropism as well as immunological parameters between virulent and classical PRRSV strains and propose a first approximation to the criteria to define a virulent PRRSV strain.

Up-Regulation of Immune Checkpoints in the Thymus of PRRSV-1-Infected Piglets in a Virulence-Dependent Fashion.

Virulent porcine reproductive and respiratory syndrome virus (PRRSV) strains, such as the Lena strain, have demonstrated a higher thymus tropism than low virulent strains. Virulent PRRSV strains lead to severe thymus atrophy, which could be related to marked immune dysregulation. Impairment of T-cell functions through immune checkpoints has been postulated as a strategy executed by PRRSV to subvert the immune response, however, its role in the thymus, a primary lymphoid organ, has not been studied yet. Therefore, the goal of this study was to evaluate the expression of selected immune checkpoints (PD1/PDL1, CTLA4, TIM3, LAG3, CD200R1 and IDO1) in the thymus of piglets infected with two different PRRSV-1 strains. Thymus samples from piglets infected with the low virulent 3249 strain, the virulent Lena strain and mock-infected were collected at 1, 3, 6, 8 and 13 days post-infection (dpi) to analyze PRRSV viral load, relative quantification and immunohistochemical staining of immune checkpoints. PD1/PDL1, CTLA4, TIM3, LAG3 and IDO1 immune checkpoints were significantly up-regulated in the thymus of PRRSV infected piglets, especially in those infected with the virulent Lena strain from 6 dpi onwards. This up-regulation was associated with disease progression, high viral load and cell death. Co-expression of these molecules can affect T-cell development, maturation and selection, negatively regulating the host immune response against PRRSV.

Chimeric HP-PRRSV2 containing an ORF2-6 consensus sequence induces antibodies with broadly neutralizing activity and confers cross protection against virulent NADC30-like isolate.

Due to the substantial genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV), commercial PRRS vaccines fail to provide sufficient cross protection. Previous studies have confirmed the existence of PRRSV broadly neutralizing antibodies (bnAbs). However, bnAbs are rarely induced by either natural infection or vaccination. In this study, we designed and synthesized a consensus sequence of PRRSV2 ORF2-6 genes (ORF2-6-CON) encoding all envelope proteins based on 30 representative Chinese PRRSV isolates. The ORF2-6-CON sequence shared > 90% nucleotide identities to all four lineages of PRRSV2 isolates in China. A chimeric virus (rJS-ORF2-6-CON) containing the ORF2-6-CON was generated using the avirulent HP-PRRSV2 JSTZ1712-12 infectious clone as a backbone. The rJS-ORF2-6-CON has similar replication efficiency as the backbone virus in vitro. Furthermore, pig inoculation and challenge studies showed that rJS-ORF2-6-CON is not pathogenic to piglets and confers better cross protection against the virulent NADC30-like isolate than a commercial HP-PRRS modified live virus (MLV) vaccine. Noticeably, the rJS-ORF2-6-CON strain could induce bnAbs while the MLV strain only induced homologous nAbs. In addition, the lineages of VDJ repertoires potentially associated with distinct nAbs were also characterized. Overall, our results demonstrate that rJS-ORF2-6-CON is a promising candidate for the development of a PRRS genetic engineered vaccine conferring cross protection.

Immunity against a Japanese local strain of porcine reproductive and respiratory syndrome virus decreases viremia and symptoms of a highly pathogenic strain.

BACKGROUND: The type 2 highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has spread throughout countries of southeast Asia, where it has caused severe economic losses. Even countries presently free of PRRSV are at high risk for infection and spread of this virus. Some of these countries, including Japan, have broad epidemics of the local type 2 PRRSV, creating chronic pathogenicity in the domestic pig population. The present study aimed to evaluate the protective efficacy of immunity by infection with a Japanese field isolate, EDRD1, against heterologous challenge with a Vietnamese HP-PRRSV field strain. To this end, four groups of PRRSV-negative crossbreed piglets were used for a challenge study. Groups 1 and 2 were inoculated with EDRD1 via the intranasal route. After 26 days, Groups 2 and 3 were inoculated with HP-PRRSV via the same route. Group 4 served as an uninfected control. Blood and oral fluid samples were taken every 3-4 days after HP-PRRSV challenge; on day 16 post-challenge, all pigs were euthanized, and examined pathologically. RESULTS: The nucleotide sequence analysis of nonstructural protein 2 gene of EDRD1 and comparison with Vietnamese HP-PRRSV showed that the 39 amino acid deletion sites of EDRD1 was nearly in the same region as the 29 amino acid deletion sites of HP-PRRSV. Immunity conferred by inoculation with EDRD1 dramatically reduced viral load in the sera and tissues besides viral shedding (Group 2) compared with those in pigs infected only with HP-PRRSV (Group 3). The clinical signs and rectal temperature were significantly reduced, and the average daily weight gain was significantly improved in the EDRD1-inoculated pigs (Group 2) compared with the Group 3 pigs. Notably, no viral RNA was detected in various organs of the Group 2 pigs 16 days post-infection with HP-PRRSV, except in one pig. Therefore, the immunity induced by EDRD1 and its genetically close field isolates may play a role in reducing viremia caused by HP-PRRSV. CONCLUSIONS: The results of the present study demonstrate that pigs are highly protected against heterologous Vietnamese HP-PRRSV challenge by immunity against a Japanese local strain, EDRD1.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) induces IL-6 production through TAK-1/JNK/AP-1 and TAK-1/NF-κB signaling pathways.

Porcine reproductive and respiratory syndrome virus (PRRSV) mainly infects monocyte/macrophage lineage and regulates the production of cytokines to influence host immune responses. Interleukin-6 (IL-6) is originally identified as a B-cell stimulatory factor and has important functions in regulating immune response, hemopoiesis, and inflammation. In this study, we verified that highly pathogenic PRRSV (HP-PRRSV) infection up-regulated IL-6 production in vivo and in vitro. Subsequently, we demonstrated that HP-PRRSV infection activated JNK and NF-κB signaling pathways to enhance IL-6 expression. We further showed that TAK-1 was important in the activation of JNK and NF-κB pathways following HP-PRRSV infection. Moreover, AP-1 and NF-κB binding motifs were found in the cloned porcine IL-6 (pIL-6) promoter, and deletion of these motifs abrogated the activation of pIL-6 promoter by HP-PRRSV, suggesting that IL-6 expression is dependent on AP-1 and NF-κB activation. These findings imply that IL-6 induced by HP-PRRSV infection is dependent on the activation of TAK-1/JNK/AP-1 and TAK-1/NF-κB signaling pathways.

Rutin, α-tocopherol, and l-ascorbic acid up-regulate type I interferon-regulated gene and type I and II interferon expressions and reduce inflammatory cytokine expressions in monocyte-derived macrophages infected with highly pathogenic porcine reproductive and respiratory syndrome virus.

This study evaluated the immunomodulatory effect of two types of phytochemicals, i.e. rutin and ß-carotene, and two types of vitamins, i.e. α-tocopherol and l-ascorbic acid on improving innate immune responses to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Monocyte-derived macrophages (MDM) from eight PRRSV-seronegative pigs were inoculated with HP-PRRSV and subsequently stimulated with rutin, ß-carotene, α-tocopherol, and l-ascorbic acid in the absence or presence of either polyinosinic:polycytidylic acid or lipopolysaccharide. The mRNA expression levels of myxovirus resistance 1, interferon regulatory factor 3 (IRF3), IRF7, 2'-5'-oligoadenylatesynthetase 1, stimulator of interferon genes (STING), osteopontin (OPN), interferon alpha (IFNα), IFNß, IFNγ, interleukin-10 (IL-10), tumor necrosis factor alpha (TNFα), and transforming growth factor beta (TGFß) were evaluated by real-time PCR. Compared with control MDM, HP-PRRSV significantly suppressed mRNA expressions of all immune-related genes except IL-10 and TGFß. Compared with HP-PRRSV-inoculated MDM, stimulation with rutin, α-tocopherol, and l-ascorbic acid, but not ß-carotene significantly enhanced mRNA expression levels of IRF3, IRF7, STING, OPN, IFNα, IFNß, and IFNγ in HP-PRRSV-inoculated MDM. Stimulation with rutin also significantly reduced mRNA expression levels of TNFα and TGFß, whereas stimulation with ß-carotene and α-tocopherol significantly reduced TNFα mRNA expression in HP-PRRSV-inoculated MDM. Our findings demonstrate the potentials of rutin, α-tocopherol, and l-ascorbic acid in enhancing type I interferon-regulated genes and type I and II IFN expressions, and in reducing pro- and/or anti-inflammatory cytokine expressions in HP-PRRSV-inoculated MDM. Our findings suggest that rutin, α-tocopherol, and l-ascorbic acid may serve as effective immunomodulators for improving innate immune response to HP-PRRSV.

Gut microbiome associations with outcome following co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in pigs immunized with a PRRS modified live virus vaccine.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most significant pathogens affecting swine. Co-infections are common and result in respiratory disease and reduced weight gain in growing pigs. Although PRRS modified live virus (MLV) vaccines are widely used to decrease PRRS-associated losses, they are generally considered inadequate for disease control. The gut microbiome provides an alternative strategy to enhance vaccine efficacy and improve PRRS control. The objective of this study was to identify gut microbiome characteristics associated with improved outcome in pigs immunized with a PRRS MLV and co-challenged with PRRSV and PCV2b. Twenty-eight days after vaccination and prior to co-challenge, fecal samples were collected from an experimental population of 50 nursery pigs. At 42 days post-challenge, 20 pigs were retrospectively identified as having high or low growth outcomes during the post-challenge period. Gut microbiomes of the two outcome groups were compared using the Lawrence Livermore Microbial Detection Array (LLMDA) and 16S rDNA sequencing. High growth outcomes were associated with several gut microbiome characteristics, such as increased bacterial diversity, increased Bacteroides pectinophilus, decreased Mycoplasmataceae species diversity, higher Firmicutes:Bacteroidetes ratios, increased relative abundance of the phylum Spirochaetes, reduced relative abundance of the family Lachnospiraceae, and increased Lachnospiraceae species C6A11 and P6B14. Overall, this study identifies gut microbiomes associated with improved outcomes in PRRS vaccinated pigs following a polymicrobial respiratory challenge and provides evidence towards the gut microbiome playing a role in PRRS vaccine efficacy.

Characterization of four types of MLV-derived porcine reproductive and respiratory syndrome viruses isolated in unvaccinated pigs from 2016 to 2020.

Modified live vaccines (MLVs) have been utilized to combat porcine reproductive and respiratory syndrome (PRRS), which raises a serious concern about the MLV-derived PRRS virus (PRRSV) isolates. During the routine investigation of PRRSV in China, four lung samples collected from unvaccinated diseased pigs from 2016 to 2020 were detected as PRRSV positive. The PRRSVs shared high ORF5 identities to CH-1R, JXA1-R, TJM-F92 and RespPRRS MLV vaccines, respectively. The viruses were isolated in Marc-145 cells and denominated as SD1612-1, JS1703-21, JSTZ1907-714 and JSYC20-05-1. Genome comparison confirmed that these isolates share the highest genomic homologies to CH-1R (97.96%), JXA1-R (99.64%), TJM-F92 (99.00%) and RespPRRS MLV (99.57%) than any other known isolates. Genome-based phylogenetic analysis showed that SD1612-1 and CH-1R, JS1703-21 and JXA1-R, JSTZ1907-714 and TJM-F92, JSYC20-05-1 and RespPRRS MLV were grouped in the same branches. In addition, amino acids unique to corresponding vaccine attenuations were also identified in our isolates. Noticeably, amino-acids potentially associated with the virulence revision from MLV strains to parental virulent viruses were also identified in the MLV-derived isolates. Our results confirm that the four types of MLV-derived isolates are circulating and evolving in Chinese swine herds for years, which highlights the necessity for the fair use of PRRS MLVs.

Upregulation of HMGB1 secretion in lungs of pigs infected by highly pathogenic porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) remains a major driver for substantial economic losses to the swine industry across the world. Pulmonary inflammatory injury is a common manifestation in infected pigs. Previous studies reported that PRRS virus (PRRSV) induces secretion of high mobility group box 1 (HMGB1), a proinflammatory factor, in cultured cells. The objective of this study was to evaluate whether HMGB1 secretion is associated with PRRSV-induced pulmonary inflammatory responses in the early stage of infection in vivo. Three-week-old piglets were inoculated with either HuN4, a highly pathogenic PRRSV (HP-PRRSV) strain, or CH1R, an avirulent PRRSV vaccine strain. Necropsy was performed at 7 days post-infection. The results showed that HuN4 significantly induced the secretion of HMGB1 and inflammatory cytokines (IL-1ß, IL-6) into the bronchoalveolar lavage fluid (BALF). HuN4 infection induced severe interstitial pneumonia in the pigs. In contrast, pigs infected by CH1R had mild lung inflammation with minimal HMGB1 secretion. In addition, high viral load of HuN4 was detected in both pulmonary alveolar macrophages (PAMs) and lung tissue, whereas viral RNA of CH1R was confined to PAMs. In consistent with the pneumonia development, HuN4 induced inflammatory cytokines in both PAMs and lung tissue, while their expression in CH1R-infected pigs confined only to PAMs. These results indicate that the HuN4-induced HMGB1 secretion into BALF may enhance the pulmonary inflammatory response and exacerbate the lung injury. This finding provides insights to the inflammatory response and pathogenesis of the HP-PRRSV infection.

An Outbreak of a Respiratory Disorder at a Russian Swine Farm Associated with the Co-Circulation of PRRSV1 and PRRSV2.

We conducted a cross-sectional study to identify the major respiratory pathogen responsible for an outbreak of respiratory disease at a swine farm in West Siberia in 2019. We discovered that the peak of morbidity and mortality coincided with a high level of porcine reproductive and respiratory syndrome virus (PRRSV) 1 and 2-related viremia. Based on longer PRRSV2 viremia, the dominant role of PRRSV2 over PRRSV1 in the outbreak was assumed. Phylogenetic analysis revealed that the PRRSV1 strain belonged to sub-genotype 2-one of the predominant groups of genotype 1 PRRSVs in Russia. A partial open reading frame 7 sequence of the PRRSV2 isolate demonstrated a high identity with modified live vaccine-related strains from Denmark (93%) and wild-type VR2332 (92%). We identified the first instance of PRRSV1/PRRSV2 mixed infection in Russia. This finding indicates that further field investigations are needed to access PRRSV2 epidemiology in eastern Europe.

Histopathological Lesions Accompanied with First-Time Isolation of a PRRSV-2 Strain in Greece.

Genotype 2 strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2) have been reported sporadically in Europe. Even if, PRRSV-2 reported to be genetically homogenous in Europe due to the introduction of an MLV vaccine strain, independent introductions of PRRSV-2 field strains have been reported. The aim of the present study was to report the complete genome sequence and evaluate the histopathological lesions of a PRRSV-2 strain, isolated for the first time in Greece. During a routine blood sampling in a commercial pig farm, the results revealed positive samples in weaners of 40-60 days for the PRRSV-2, using real-time polymerase chain reaction. The clinical picture was characterized from respiratory symptoms in weaners, as well as coughing and poor performance at finishing stage and less than 3% mortality rate from weaning stage to finishing stage. The use of ORF5 for PRRSV phylogenetic analysis of the isolated PRRSV strain, named "x1544-1 strain", was successfully determined, belonging to the genotype PRRSV-2. Comparison of the obtained sequence revealed nucleotide sequence identity >98% with PRRSV-2 strain VR2332 and other related strains from Denmark and China. The histopathological evaluation revealed diffuse interstitial pneumonia, multifocal interstitial nephritis, while in the lymphoid organs, follicular and paracortical hyperplasia, coexisting with necrosis and depletion of germ cells were detected. The results of current study undersign the importance for veterinary practitioners to have up-to-date access to phylogenetic data linked to phenotypic information to follow-up the control and prevention strategies against PRRSV.

Elastase-mediated membrane fusion of highly pathogenic porcine reproductive and respiratory syndrome virus at host cell surface.

Infection by enveloped viruses includes endocytosis and/or membrane fusion at the plasma membrane, where host cell proteases play an essential role. Among them, elastase-mediated infection has been documented for several enveloped viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically critical factor in global swine industry, is previously reported to infect host cells via low pH-dependent clathrin-mediated endocytosis (CME) and undergo membrane fusion in recycling endosomes. In the current study, we identified that elastase was significantly elevated in the lung tissues of highly pathogenic PRRSV (HP-PRRSV)-infected pigs compared to the mock-infected ones. We subsequently demonstrated that elastase contributed to HP-PRRSV infection in both MARC-145 cells and porcine alveolar macrophages (PAMs). Mechanistically, HP-PRRSV entered host cells at the cell surface via elastase-mediated membrane fusion, independent of low pH and CME, and its glycoprotein 5 (GP5) was cleaved by the protease during this process. All these findings deepen our understanding of HP-PRRSV infection, and are beneficial for prevention and control of the disease.

CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance.

Porcine reproductive and respiratory syndrome virus (PRRSV) and transmissible gastroenteritis virus (TGEV) are two highly infectious and lethal viruses causing major economic losses to pig production. Here, we report generation of double-gene-knockout (DKO) pigs harboring edited knockout alleles for known receptor proteins CD163 and pAPN and show that DKO pigs are completely resistant to genotype 2 PRRSV and TGEV. We found no differences in meat-production or reproductive-performance traits between wild-type and DKO pigs, but detected increased iron in DKO muscle. Additional infection challenge experiments showed that DKO pigs exhibited decreased susceptibility to porcine deltacoronavirus (PDCoV), thus offering unprecedented in vivo evidence of pAPN as one of PDCoV receptors. Beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit pAPN or CD163 for entry.

Pig epidemics are the biggest threat to the pork industry. In 2019 alone, hundreds of billions of dollars worldwide were lost due to various pig diseases, many of them caused by viruses. The porcine reproductive and respiratory virus (PRRS virus for short), for instance, leads to reproductive disorders such as stillbirths and premature labor. Two coronaviruses ­ the transmissible gastroenteritis virus (or TGEV) and the porcine delta coronavirus ­ cause deadly diarrhea and could potentially cross over into humans. Unfortunately, there are still no safe and effective methods to prevent or control these pig illnesses, but growing disease-resistant pigs could reduce both financial and animal losses. Traditionally, breeding pigs to have a particular trait is a slow process that can take many years. But with gene editing technology, it is possible to change or remove specific genes in a single generation of animals. When viruses infect a host, they use certain proteins on the surface of the host's cells to find their inside: the PRRS virus relies a protein called CD163, and TGEV uses pAPN. Xu, Zhou, Mu et al. used gene editing technology to delete the genes that encode the CD163 and pAPN proteins in pigs. When the animals were infected with PRRS virus or TGEV, the non-edited pigs got sick but the gene-edited animals remained healthy. Unexpectedly, pigs without CD163 and pAPN also coped better with porcine delta coronavirus infections, suggesting that CD163 and pAPN may also help this coronavirus infect cells. Finally, the gene-edited pigs reproduced and produced meat as well as the control pigs. These experiments show that gene editing can be a powerful technology for producing animals with desirable traits. The gene-edited pigs also provide new knowledge about how porcine viruses infect pigs, and may offer a starting point to breed disease-resistant animals on a larger scale.